Salt retention is generally regarded as the most common cause of high blood pressure (BP) in humans. One in three US adults has high BP (hypertension), which can lead to more serious complications such as heart failure and stroke. Therefore, it is important to understand the mechanisms that link salt retention to hypertension in humans in an effort to better control, or even prevent this disease. Recent studies have identified several key steps in the pathway linking salt to hypertension in rodents. First, salt retention promotes secretion of the adrenocortical hormone, endogenous ouabain (EO). EO then specifically binds and inhibits arterial smooth muscle cell (ASMC) ouabain-sensitive a2 Na+ pumps, while the more abundant ouabain-resistant a1 pumps are unaffected. The a2 Na+ pumps localize specifically to plasma membrane (PM) microdomains overlying the sarcoplasmic reticulum (SR);thus ouabain increases only the local Na+ concentration in the tiny "junctional spaces" between the PM and SR. The exchange of cell Na+ for extracellular Ca2+ by the Na+/Ca2+ exchanger-1 (NCX1), which is also located at PM-SR junctions, increases Ca2+ retention. This in turn raises vascular tone and peripheral vascular resistance, and thereby elevates BP. Parallels of the rodent mechanism can be seen in humans. One major issue, however, with translating the rodent mechanism to humans is that the predominant ("housekeeping") a1 Na+ pumps in humans are ouabain-sensitive. Therefore, it is not clear whether the a1 and/or a2 Na+ pumps are involved in ouabain-dependent BP increases in humans. My goal here is to test the hypothesis that in human ASMCs (hASMCs), inhibition of a2 Na+ pumps, and not a1 Na+ pumps, by nanomolar ouabain augments Ca2+ signaling, as in rodent ASMCs. I will test this hypothesis by first determining what Na+ pump a (catalytic) subunit isoforms are expressed in hASMCs and what is their relative abundance (Aim 1). Next, I will determine how the Na+ pump a isoforms are localized/distributed in hASMC PM by performing high-resolution confocal immunocytochemistry on primary hASMCs (Aim 2). Finally, I will determine whether low dose (1-10 nM) ouabain augments Ca2+ signaling in response to vasoconstrictors (serotonin, ATP, and high K+) using fura-2 (Aim 3). If augmentation is seen, I will determine whether this effect is due to a1 or a2 Na+ pumps by lipofectamine transfection of hASMCs with either an ouabain-resistant (OR) or dominant negative (DN) rat a2 Na+ pump construct. Verification of my hypothesis via these experiments will provide the first direct evidence that key molecular mechanisms that underlie salt-dependent hypertension in rodents are present and function similarly in humans. PUBLIC HEALTH RELEVANCE: The goal of this project is to determine whether the specific mechanisms that link excess salt to elevated blood pressure in rodents also exist in humans. I plan to achieve this goal by testing for the identified components of the rodent mechanism in human arterial cells. I anticipate that the results will provide the first direct evidence that the underlying key molecular mechanisms in salt-dependent hypertension in rodents function similarly in humans.